Departments of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates University Al Ain, UAE.

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Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University Al Ain, UAE.

Abstract

The cannabinoid type two receptors (CB2), an important component of the endocannabinoid system, have recently emerged as neuromodulators and therapeutic targets for neurodegenerative diseases including Parkinson's disease (PD). The downregulation of CB2 receptors has been reported in the brains of PD patients. Therefore, both the activation and the upregulation of the CB2 receptors are believed to protect against the neurodegenerative changes in PD. In the present study, we investigated the CB2 receptor-mediated neuroprotective effect of β-caryophyllene (BCP), a naturally occurring CB2 receptor agonist, in, a clinically relevant, rotenone (ROT)-induced animal model of PD. ROT (2.5 mg/kg BW) was injected intraperitoneally (i.p.) once daily for 4 weeks to induce PD in male Wistar rats. ROT injections induced a significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and DA striatal fibers, following activation of glial cells (astrocytes and microglia). ROT also caused oxidative injury evidenced by the loss of antioxidant enzymes and increased nitrite levels, and induction of proinflammatory cytokines: IL-1β, IL-6 and TNF-α, as well as inflammatory mediators: NF-κB, COX-2, and iNOS. However, treatment with BCP attenuated induction of proinflammatory cytokines and inflammatory mediators in ROT-challenged rats. BCP supplementation also prevented depletion of glutathione concomitant to reduced lipid peroxidation and augmentation of antioxidant enzymes: SOD and catalase. The results were further supported by tyrosine hydroxylase immunohistochemistry, which illustrated the rescue of the DA neurons and fibers subsequent to reduced activation of glial cells. Interestingly, BCP supplementation demonstrated the potent therapeutic effects against ROT-induced neurodegeneration, which was evidenced by BCP-mediated CB2 receptor activation and the fact that, prior administration of the CB2 receptor antagonist AM630 diminished the beneficial effects of BCP. The present study suggests that BCP has the potential therapeutic efficacy to elicit significant neuroprotection by its anti-inflammatory and antioxidant activities mediated by activation of the CB2 receptors.

The expression level of cannabinoid receptor 2 (CB2) measured by western blot in the striatum. (A) The western blot showing CB2 expression. (B) CB2 expression was decreased in the ROT group as compared to the CONT group rats. However, BCP supplementation of ROT injected rats showed increased expression of CB2 receptors. Selective antagonist of CB2 receptors, AM630 administration prior to BCP supplementation decreased the expression of CB2 receptors. AM630 administration to ROT injected rats has not shown any noticeable difference in the expression of CB2. Finally, AM630 alone had no effect on the level of CB2 expression as compared to control rats. Values are expressed as percent (%) mean ± SEM (n = 3).

The immunohistochemistry for tyrosine hydroxylase (TH) to detect the expression of the TH-immunoreactive (TH-ir) dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the TH-ir dopamine nerve fibers in the striatum. Values are expressed as percent mean ± SEM (n = 3). The scale bar is 100 μm. (A) The number of TH-ir neurons was decreased in the SNpc of ROT-injected rats as compared to control (CONT) group. While, BCP supplementation leads to increased expression of TH-ir neurons in ROT+BCP group as compared to ROT group. Furthermore, the ROT-BCP+AM630 group showed decreased expression of TH-ir neurons as compared to ROT+BCP group. (B) Similarly, TH-ir DA nerve fibers immunofluorescence was also decreased in the ROT group in comparison to the CONT group rats. Moreover, BCP supplementation increased the immunoreactivity of DA fibers as compared to ROT group. The ROT-BCP+AM630 group showed TH-ir DA fibers expression similar to ROT group. (C,D) The number of TH-ir DA neurons in the SNpc was counted from each group. A significant (*p < 0.05) decrease in the number of DA neurons was observed in the SNpc of the ROT group when compared to the CONT group. BCP treatment to ROT administered rats significantly (#p < 0.05) protected the DA neurons from the ROT-induced neuronal death in the ROT+BCP group. The ROT-BCP+AM630 group also showed significantly (δp < 0.05) decreased number of DA neurons as compared to the ROT+BCP group. No significant differences were observed between the DA neurons of the CONT and AM630-only group. (E) Immunofluorescence of the TH-ir DA fibers was observed in the striatum of different groups. Significant (*p < 0.05) decrease in the TH-ir striatal DA fibers was observed in the ROT group as compared to the CONT group. Although, BCP supplementation of ROT treated rats significantly (#p < 0.05) inhibited the loss of the TH-ir DA fibers in the ROT+BCP group as compared to the ROT group. ROT-BCP+AM630 group showed significant (δp < 0.05) loss of the TH-ir DA fibers when compared to the ROT+BCP group. CONT and AM630-only group did not show any apparent loss of TH-ir DA fibers.

The immunofluorescence staining to detect the expression of glial fibrillary acidic protein (GFAP) positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba-1) positive microglia in the striatum. Values are expressed as percent mean ± SEM (n = 3). The scale bar is 200 μm. Profound expression of GFAP positive astrocytes (A) and Iba-1 positive microglia (B) was found in the ROT-administered and ROT-BCP+AM630 groups as compared to the vehicle-injected CONT and AM630-only groups. While BCP supplementation to ROT-injected rats showed moderate staining of GFAP and Iba-1 as compared to ROT-injected rats. Quantitative analysis of activated astrocytes (C) and microglia (D) has shown that significant (*p < 0.05) increase in number of activated astrocytes and microglia was observed in the ROT group as compared to the CONT group. However, BCP supplementation significantly (#p < 0.05) reduced the number of activated astrocytes and microglia in the ROT+BCP group as compared to the ROT group. The ROT-BCP+AM630 group also showed significantly (δp < 0.05) increased number of activated astrocytes and microglia when compared to the ROT+BCP group. The CONT and AM630-only groups did not show any marked difference in the activation of astrocytes and microglia.

The pro-inflammatory cytokines: IL-1β, IL-6, and TNF-α were measured by enzyme linked immunosorbent assay (ELISA) in the midbrain. Values are expressed as mean ± SEM (n = 6–8). The level of IL-1β (A), IL-6 (B), and TNF-α (C) was significantly (*p < 0.05) increased in the ROT group when compared to the CONT group. Although, BCP supplementation significantly (#p < 0.05) decreased the ROT-induced increase of these proinflammatory cytokines in the ROT+BCP group. The ROT-BCP+AM630 group showed significantly (δp < 0.05) increased level of IL-1β, IL-6, and TNF-α as compared to the ROT+BCP group. There is no significant difference in these cytokines between the CONT and the AM630-only group.

Western blot analysis of NF-κB p65, COX-2, and iNOS in the striatal tissue. Values are expressed as percent (%) mean ± SEM (n = 3). Expression level of NF-κB p65 (A) COX-2 and iNOS (B) was carried out by western blot. (C) ROT-administered group rats showed increase in nuclear NF-κB p65 level as compared to control group (171.7 vs. 100% control). BCP treatment of the ROT-challenged rats decreased the NF-κB p65 expression when compared to the ROT group (112.38 vs. 171.7%). The ROT-BCP+AM630 group had shown increased expression of NF-κB p65 as compared to the ROT+BCP group (171.55 vs. 112.38%). (D) ROT-injected rats showed increased COX-2 level as compared to the CONT rats (213.48 vs. 100% control). BCP supplementation to ROT-injected rats showed decreased expression of COX-2 as compared to ROT group rats (143.6 vs. 213.48%). ROT-BCP+AM630 group rats showed increase in COX-2 expression as compared to the ROT+BCP group rats (205.6 vs. 143.6%). (E) Likewise, iNOS expression was increased in the ROT group rats as compared to the CONT group rats (251.56 vs. 100% control). BCP treatment decreased iNOS expression as compared to the ROT group rats (158.12 vs. 251.56%). The ROT-BCP+AM630 group showed increase in iNOS expression when compared to the ROT+BCP group (247.18 vs. 158.12%).